Dopamine provides reward for adaptive behaviors such as food and sexual activity, but drugs of[unreadable] abuse release dopamine directly, bypassing the normal regulatory mechanisms and producing long-term[unreadable] changes that result in tolerance, physical dependence and drug craving. The long-term objective of this[unreadable] proposal is to understand how dopamine release is regulated, and how changes in release influence[unreadable] behavior and contribute to drug abuse. The strategy is to use vesicular monoamine transporter 2[unreadable] (VMAT2) as a tool to characterize the exocytotic release of monoamines, and to study it as a locus for[unreadable] regulation.[unreadable] Unlike most other classical neurotransmitters, monoamines including dopamine undergo release[unreadable] from cell body and dendrites as well as the axpn terminal. Importantly, somatodendritic dopamine[unreadable] release is required to induce behavioral sensitization, a model for drug-seeking. However, the[unreadable] mechanism of somatodendritic dopamine release remains unclear, with some evidence favoring vesicular[unreadable] release and other reverse flux through the dopamine transporter. In Specific Aim 1, we will assess the[unreadable] potential for exocytotic release by studying the trafficking of VMAT2, the carrier required for loading[unreadable] vesicles with monoamine. Preliminary data suggests that somatodendritic vesicles containing VMAT2[unreadable] undergo regulated exocytosis, and we will now use both fixed cells and live cell imaging to characterize[unreadable] the exocytosis and endocytosis of VMAT2 in live neurons, and to identify the signals responsible for[unreadable] sorting VMAT2 to this pathway. We will then use this information to manipulate the localization of VMAT2[unreadable] in vivo, test unambiguously the mechanism of somatodendritic dopamine release, and determine the role[unreadable] of somatodendritic release in the plasticity of monoamine systems in general.[unreadable] In Specific Aim 2, we will use VMAT2 to address basic questions about the relationship between[unreadable] vesicle filling and the recycling of membrane in the synaptic vesicle cycle. Exploring the biochemical[unreadable] basis for a genetic interaction identified in C. elegans, we have found that the v-SNARE synaptobrevin[unreadable] required for regulated exocytosis inhibits the activity of VMAT2, and this effect does not reflect changes in[unreadable] transporter expression or trafficking. Rather, the v-SNARE appears to inhibit VMAT2 directly. We will[unreadable] now characterize the mechanism for this inhibition, and use this information to determine the role of this[unreadable] interaction in dopamine release. The results will provide physiologically relevant information about a[unreadable] novel mechanism regulating vesicular neurotransmitter transport that may contribute to the long-term[unreadable] changes in dopamine release that accompany behavioral sensitization.